Impacts of thyme and/or garlic oils on growth, immunity, antioxidant and net farm income in Damascus goats

This study aimed to evaluate the impact of thyme and/or garlic oil administration on growth performance, immunity, antioxidant, biochemical parameters, and net farm income of Damascus goats. Forty weaned Damascus goats were allocated into four groups. The first group was the control without oral administration, while the 2nd (Th), 3rd (Gr), and 4th (ThGr) groups were orally administrated by (2 ml/goat/day) of thyme oil, garlic oil and their mixture (1:1), respectively during the whole experiment period. The final body weight of goats orally administered oil mixture was the heaviest group, it was 10, 4.5 and 3.5% than the control, Th. and Gr. groups, respectively with better feed conversion ratio and high net farm income. Goats of ThGr. group revealed the best immunity, antioxidant and general health condition than the control group with 50% reduction of MDA. Liver (AST, 33% and ALT, 38%) and kidney (creatinine, 88%) functions improved by oils mixtures orally administration compared with the control group. LDL, triglyceride and cholesterol were reduced by 47, 33 and 21% compared with the control group, respectively. Thus, mixture oil administration (thyme and garlic at the ratio of 1:1, 2 ml/goat/day) improved growth (10%), antioxidant status (MDA 50%), liver (AST, 33% and ALT, 38%), kidney function (creatinine, 88%), the FCR (17.4%) and net farm income (21%), of Damascus goats.


Materials and methods
All experimental procedures were accomplished with reference to the local Experimental Animal Care Committee and approved (20209) by the Institutional Committee of the Department of Animal Production, Faculty of Agriculture, Benha University, Egypt.

Diet, experimental design and management
This study was carried out for three months (February to April 2021) at the experimental farm of the Faculty of Agriculture, Benha University, Egypt with 40 weaned female Shami goats (Damascus) with 17.86 ± 0.22 kg average body weight and 6-8 months of age.Goats were fed on a basal diet and classified to four groups (10 goats in each group), the first group was the control (cont; without any oral administration); the second (Th), third (Gr) and fourth group (ThGr) were orally administrated by thyme oil (2 ml/goat/day), garlic oil (2 ml/goat/day) and mixed oil (1 ml thyme + 1 ml garlic/goat/day), respectively just before feeding during the whole experiment period (3 months).Goats were housed in individual pens (0.97 × 2.82 m) bedded with straw.
All experimental goats were individually weighed at the beginning of the experiment and every month until the end of the experiment to the nearest 10 g.The average daily gain (ADG) was calculated using this formula: ADG = Final body weight (kg) − Initial body weight (kg)/experimental duration (days).Both consumed diets and refusals (if any) were recorded daily.Total and daily feed intake (TFI, DFI) and feed conversion ratio (FCR) were then calculated.Feed conversion ratio (FCR) = Total feed intake (kg)/total weight gain (kg).Goats were housed individually in hygienic metallic pens and supplied with clean water all time.The routine vaccination program against infectious diseases and medications against internal and external parasites were applied.
The basal diet was formulated according to the recommendations of NRC 32 (Table 1).The main components of the thyme and garlic essential oil were represented in Tables 2 and 3, respectively).A Hewlett-Packard II 5890 gas chromatography (GC) system with an FID detector and an HP-5 ms capillary column (30 m × 0.25 mm, film thickness 0.25 m) was used to analyze the essential oils.The temperatures of the injector and the detector were set to 290 °C and 220 °C, respectively.The GC stove temperature was raised from 60 to 240 °C at 3 °C min −1 and held isothermally for 10 min.At a flowrate of 1 ml min −1 , helium served as the carrier gas.Samples diluted to 1:100 in diethyl ether by volume; in the spitless mode were manually injected.Quantitative information was acquired electronically from FID region rate information without the utilization of amendment factors.Individual component relative percentages expressed as percent peak area compared to the total composition of the EO as determined by GC-MS analysis 33,34 .

Net farm income
Finance analysis has been done to assess and compare the profitability among the various groups.The net farm income (NFI) was assessed 37 from the difference between total income (TI) and total costs (TC).TI and TC were individually assessed for each goat.TC include the feeding costs (diet plus oral oils), the labor costs (the price farmers' working hour multiplied by the number of hours employed per goat 38 , veterinary care costs (vaccines, and veterinary supervision).Water, electricity, and equipment maintenance in addition to costs of litter and building rent value were also calculated per goat for each group.TI consisted of income value from marketable goats' body weight gain.All these variables were calculated in Dollars.

Blood sampling and analysis
Blood samples were collected at the end of the experiment from all experimental goats in the morning just preceding feeding from the jugular vein to a clean dry test tube.The samples were left at room temperature for 45-65 min and then centrifuged for 30 min at 4000 rpm.Serum was separated into clean dried glass vials (5-7 ml) and stored frozen (− 20 °C) until analysis.Blood was analyzed for total protein 39 , albumin 40 , globulin Table 1.Goats' diet with its chemical composition.a Each 1 kg of vitamins and minerals mixture contains: Vit.A = 4,000,000 IU, Vit.D 3 = 833,333.33IU, Vit.E = 5000 mg, Zn = 20,000 mg, Mg = 23,333.33mg, Fe = 20,000 mg, Cu = 10,000 mg, I = 1666.66mg, Se = 100 mg, Co = 333.33   www.nature.com/scientificreports/(by subtracting the albumin value from the total protein concentration), urea 41 , creatinine, ALT and AST 42 with commercial kits (Wako Pure Chemical Industries, Osaka, Japan).Single radial immune diffusion technique was used to quantify total immunoglobulin IgA, IgM, IgG in blood serum (bind ARIDtm Blinding site limited, Birmingham, UK) according to the method described by Fahey and McKelvey 43 .Serum malondialdehyde (MDA) level was determined 44 .Serum Superoxide Dismutase (SOD) activity was assayed at 420 nm on a UV-Vis Shimadzu spectrophotometer (2450) 45 .Catalase activity (CAT) was measured by the spectrophotometric method based on the decomposition of H 2 O 2 46 .Blood LDL, HDL, triglyceride, and cholesterol were measured 47 with commercial kits (Wako Pure Chemical Industries, Osaka, Japan).

Statistical analysis
The data were analyzed using one-way analysis of variance (ANOVA) (SAS: Statistical Analysis System software version 2004).Shapiro-Wilk tests has been used to test the data normality and it was normally distributed.Duncan's multiple-range tests was used to determine the significance differences among groups.The applied static model is as follows: Where yij is the observations, µ = general mean, Ti is the effect of i th diet supplementation, where i = 1, 2, 3, and 4 and eij is the random error.

Ethical approval
All experimental procedures were accomplished with reference to the local Experimental Animal Care Committee and approved (20209) by the Institutional Committee of the Department of Animal Production, Faculty of Agriculture, Benha University, Egypt.The current study is reported in accordance with ARRIVE guidelines.

Results
The thyme and garlic oil orally administration improved the growth performance, weight gain, average daily gain, feed conversion ratio, and net farm income.The final body weight of goats orally supplemented with oil mixture of thyme and garlic was the heaviest group.It was 10, 4.5 and 3.5% than the cont, Th. and Gr.groups, respectively.The final weight gain and the average daily gain were the best of the goats orally supplemented with mixture of garlic and thyme oil.They were 12.55 kg and 0.14 kg/day, respectively.Moreover, their feed conversion was better (17, 11 and 7%) with high net farm income (21, 11.5 and 7%) compared with the cont, Th. and Gr.groups, respectively (Table 4).
Thyme and garlic oils improved the goats' immune status with better antioxidant levels.Goats of the ThGr group revealed the best immunity, antioxidant and general health condition.IgA (21, 7 and 7%), SOD (62, 51 and 43%), CAT (27, 24 and 12%) and TAC (30, 25 and 21%) were better than the cont, Th and Gr groups, respectively with a reduction of MDA by 50, 37 and 18%, respectively.The liver and kidney functions improved by thyme and garlic oils orally supplementation.AST (33, 22 and 24%), ALT (38, 23 and 18%) and creatinine (88, 18 and 34%) levels were reduced compared with the other three groups, respectively (Table 5).The total protein and globulin were the highest in the group orally supplemented with the oil mixture compared with the other experimental groups with better HDL in Gr. and ThGr.groups.While LDL was the lowest in ThGr. group.Concurrently, the triglyceride and cholesterol were reduced by 33 and 21% compared with the cont group, respectively (Table 5).

Discussion
Thyme, garlic and their essential oils are natural alternatives that may be used for growth promoters and antibiotics in ruminant rations [6][7][8] .Essential oils can be used in dairy cows 9 .This research aimed to evaluate the potential impact of thyme and garlic oil orally administration to Damascus goats on growth performance, immunity, antioxidant, biochemical parameters and net farm income.
It is tremendously challenging to compare the investigations that used various essential oils since the outcomes will be established on several issues, such as oil composition, doses, extraction methods, route of administration,  48,49 .Zhong et al. 49 reported that garlic dietary supplementation augmented the average daily gain in lambs.Moreover, Abdel-Wareth and Metwally 48 stated that the daily weight gain and FCR of male Californian rabbits were improved by thyme supplementation without any effect on daily feed intake.The current findings did not show any significant variations among the various groups regarding total and daily feed intake supported by others in beef cattle 50,51 .There were conflicting results regarding FCR.Some authors reported that thyme and/ or garlic did not affect FCR 49,[52][53][54] , while others detect an improvement 48 .The current findings were comparable with Abdel-Wareth and Metwally 48 .The reported feed intake, ADG and FCR of the current study were similar to that performed on goats 53 .The gastrointestinal nematodes infestation of small animals reduced the average daily gain (23-63%) in lambs 55 and feed intake 56 , feed digestion in calves 57 , meat quality in lambs 58 with an increase of FCR in sheep 59 .Therefore, thyme and/or garlic supplementation reduced the parasitic load and consequently improved the growth and productive performance of treated calves than the control group 57 .Moreover, there was an increase in the goats' live weight treated with anthelmintic than in the control group.Dietary garlic oil supplementation did not affect feed intake and average daily gain of lambs without parasitic infestation 60 .While Hasan et al. 24 reported an increase in average daily gain (10.3%) of grazing goats suffered from internal parasites if they were supplemented orally with garlic solution 24 .
The current study revealed that the oil mixture increased the final body weight by 10% which was comparable to Hasan et al. 24 on grazing goats.This improvement may be attributable to several factors (a) the anthelmintic effect of the thyme and garlic oils that reduced the gastrointestinal parasites which hampering the growth of small ruminants leading to economic losses 61,62 , (b) the power antimicrobial activity against gram-positive and negative bacteria through disruption the bacterial plasma membrane and glucose uptake reduction, while the beneficial bacteria are less sensitive to the inhibitory effects of garlic 27,63,64 , (c) thyme methanolic extract may influence the fermentation efficiency of rumen and may be used as a mitigating mediator of methane 65 , (d) improved the Table 4. Effect of oral administration of thyme and/or garlic on growth performance, feed intake, feed conversion and economic feed efficiency of weaned Damascus goats.SEM Standard error mean, BW Body weight, WG Weight gain, ADG Average daily gain, TFI Total feed intake, FCR Feed conversion ratio.NFI Net farm income during the 3 months of the experimental period (Benefit/cost ratio) with 5.66 and 0.33$ for each kg of Shami goats body weight and feed intake, respectively.a,b,c Means within the same row with different superscript letters differ significantly (p ≤ 0.05).www.nature.com/scientificreports/digestibility and feed efficiency of growing lambs 66 through activation of ruminal bacteria, stimulation the secretion and flow of bile, saliva and gastric enzymes [67][68][69] that may modify the microbial numbers in the rumen and decreased the of dietary protein degradation with increasing N escape and protein flow to the small intestine, consequently improved the nitrogen utilization 70,71 , (e) increased the synthesis and activity of pancreatic enzymes, enriching digestion, absorption and utilization of proteins in the small intestine 72 , (f) reducing the proportion of acetate with increasing the propionate 73 , (g) thymol may protect the microvilli 74 .The current study revealed that ThGr groups was the best regarding the net farm income.Better performance of the animal such as weight gain and FCR will providing higher incomes, total profit margin with reducing production costs 75,76 .El-Azrak et al. 77 reported an improvement in the immune response and serum IgG following essential oil supplementation of goats which supported the current results 78 .This may be owned to the increasing phagocytosis as observed in pigs 79 .Moreover, the essential oils may be reduced the animal stress because the stress reduced the release of immunoglobulins 80 .Releasing somatostatin and adrenal corticosteroid hormones are accountable for lowering the immunoglobulins production 80 .Blood parameters are imperative tool for detecting the metabolic syndromes 81 and to estimate the physiological state of animals 82 .MDA and glutathione are the foremost marker of lipid peroxidation utilized for assessing the oxidative damage 83,84 .Antioxidant enzymes (SOD) accomplish an imperative role of antioxidant defenses 85 .Stress and high lipid peroxidation increased the production of ROS that increased oxidative destruction and MDA level with a reduction of the antioxidant enzymes activity [86][87][88] .Thymol boosts the total antioxidant condition in vivo 89 due to it improves the activity of antioxidant enzymes, such as superoxide dismutase and catalase 90 .

Parameter
Liver is an imperative organ in metabolic body processes.Serum Transaminases activities were represented the damage and recovery of the liver cells and the pathological condition 91,92 that are subtle to the toxic matters 93 .This increase can be ascribed to liver or muscle damage, septicemia, and/or toxemia and their values are associated with the severity of cell damage 94 .Aba et al. 95 stated that the normal level of goats AST (IU/l), ALT (IU/l), albumin (g/dl) and cholesterol (mg/dl) were 28.60-92, 2.59-29.65,2.75-3.86 and 33.84-132.42,respectively.The oral administration of phytobiotics enhanced the liver function of rabbits 96 which supported the current findings.Ghoneem and Mahmoud 97 revealed that there was an increase of the AST (2.5%) and ALT (10%) levels with thyme essential oil supplementation in Damascus goats.But Abdel-Wareth and Metwally 48 detected a reduction of AST and ALT in male rabbits.Moreover, Usur 53 stated that essential oil reduced AST and ALT by 8 and 11%, respectively in goats.Vakili et al. 98 reported that essential oils supplementation did not influence on hepatic enzymes 67,99,100 .
The creatinine level of the current study was comparable to that reported by Ghoneem and Mahmoud 97 .Thyme and/or garlic supplementation increased the serum creatinine and urea in goats 53,67,97 .On the other hand, this study revealed a reduction of creatinine level that supported by Abdel-Wareth and Metwally 48 in male rabbits.But others did not detect any difference 98,[101][102][103] in ewes, lambs and calves.Essential oils did not influence the serum urea level that was supported by other 77 in goats.The creatinine levels in this investigation are within the normal reported range (0.6-1.1 mg/dl) 1,104 .The current finding regarding the levels of total protein, albumin and globulin was similar to the findings of others in goats 1,53,97 .There have been conflicting results on the effect of orally supplemented mixture of thyme and garlic oil on the total protein, albumin and globulin.Several authors have detected an increase of their levels [105][106][107] .While others have stated that there were no differences following thyme and/or garlic supplementation 77,97,108,109 .The current outcomes were similar with the findings of the research that stated an increase of total protein and globulin after orally administration of thyme and/or garlic oil.
The high levels of total protein and globulin in goats treated with oil mixture may be attributable to; (a) the serum protein was positively associated with body weight and protein manufacture 110,111 and food protein 101 , (b) they stimulate liver cells to multiplication the protein synthesis 109 , (c) the improvements of ruminal microbial protein synthesis 73 and crude protein digestibility 97 .On the other hand, there was a reduction of total protein followed ginger oil supplementation due to the modification effect of essential oils on the ruminal microbial population status and decreasing the contribution of Prevotella spp. that is chiefly accountable for protein degradation and amino acids deamination and consequently, protein metabolism 112 .
There have been inconsistent outcomes on the influence of orally supplemented thyme and/or garlic on goats' cholesterol and triglycerides.Whereas several authors have reported that this supplementation reduced cholesterol and triglycerides 53,97,113,114 in goats.Others did not detect any difference 67,98,101,108,115 in calves and lambs.While others reported an increase of cholesterol and triglycerides 77 in goats.Nonetheless, these results agreed with the majority of studies that have reported a reduction cholesterol and triglycerides.
Cholesterol was responsible for the level of triglycerides in the blood.Thyme and garlic supplementation reduced cholesterol and triglycerides by 11 and 18% 97 in Damascus goats, 11 and 7%, respectively 53 in goats.While the current study revealed 21 and 33% reductions of cholesterol and triglycerides, respectively.These results may be attributed to; (a) decreasing the efficiency of enzymes or preventing the thiol enzymes such as HMGCoA 24 and CoASH 116 in liver, (b) the hypolipidemic and hypercholesterolemia characters of aromatic plants and their essential oil 117 , (c) the metabolism of herbal plants product might prevent the production of 3-hydroxy-3-methylglutaryl CO.A enzyme, which influences the cholesterol synthesis 118 .Moreover, the HDL was increased by 21% followed the orally administration of the oil mixture that may be owned to decrease the values of triglycerides, cholesterol and LDL 119 .Our finding regarding HDL was comparable with Meteab et al. 1 who reported 39.06 mg/dl of HDL in Damascus goat.

Conclusions
Recently consumers are seeking for safe animal products without synthetic chemical residues.Therefore, researchers and breeders are trying to find natural alternatives for growth promoters and antibiotics in ruminant rations as herbal plants and their essential oils.Thus, mixture oil administration (thyme and garlic at the ratio of 1:1, 2 ml/goat/day) improved growth (10%), antioxidant status (MDA 50%), liver (AST, 33% and ALT, 38%), kidney function (creatinine, 88%), the FCR (17.4%) and net farm income (21%), of Damascus goats.Therefore, this study recommended supplementing goats with the mixture of thyme and garlic oil for better growth and health.

Table 2 .
The main components of the thyme essential oil.Gas chromatography/mass spectrometry (GC-MS) was used to examine the chemical components of thyme essential oil.

Table 3 .
The main components of the garlic essential oil.Gas chromatography/mass spectrometry (GC-MS) was used to examine the chemical components of garlic essential oil.

Table 5 .
Effect of oral administration of thyme and/or garlic on serum immunity, antioxidant and biochemical parameters of weaned Damascus goats.SEM Standard error mean, MDA Malondialdehyde, CAT catalase, SOD superoxide dismutase, TAC total antioxidant capacity, IgG Immunoglobulin G, IgA Immunoglobulin A, IgM Immunoglobulin M, AST Aspartate aminotransferase, ALT Alanine aminotransferase, LDL-c low-density lipoprotein-cholesterol, HDL-c High-density lipoprotein-cholesterol. a,b,c Means within the same row with different superscript letters differ significantly (p ≤ 0.05).